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CRISPR-Cas12a/Cpf1-assisted precise, efficient and multiplexed genome-editing in Yarrowia lipolytica

CRISPR-Cas9 has been widely adopted as the basic toolkit for precise genome-editing and engineering in various organisms. Alternative to Cas9, Cas12 or Cpf1 uses a simple crRNA as a guide and expands the protospacer adjacent motif (PAM) sequence to TTTN. This unique PAM sequence of Cpf1 may signific...

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Autores principales: Yang, Zhiliang, Edwards, Harley, Xu, Peng
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Elsevier 2019
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6906711/
https://www.ncbi.nlm.nih.gov/pubmed/31867213
http://dx.doi.org/10.1016/j.mec.2019.e00112
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author Yang, Zhiliang
Edwards, Harley
Xu, Peng
author_facet Yang, Zhiliang
Edwards, Harley
Xu, Peng
author_sort Yang, Zhiliang
collection PubMed
description CRISPR-Cas9 has been widely adopted as the basic toolkit for precise genome-editing and engineering in various organisms. Alternative to Cas9, Cas12 or Cpf1 uses a simple crRNA as a guide and expands the protospacer adjacent motif (PAM) sequence to TTTN. This unique PAM sequence of Cpf1 may significantly increase the on-target editing efficiency due to lower chance of Cpf1 misreading the PAMs on a high GC genome. To demonstrate the utility of CRISPR-Cpf1, we have optimized the CRISPR-Cpf1 system and achieved high-editing efficiency for two counter-selectable markers in the industrially-relevant oleaginous yeast Yarrowia lipolytica: arginine permease (93% for CAN1) and orotidine 5′-phosphate decarboxylase (~96% for URA3). Both mutations were validated by indel mutation sequencing. For the first time, we further expanded this toolkit to edit three sulfur house-keeping genetic markers (40%–75% for MET2, MET6 and MET25), which confers yeast distinct colony color changes due to the formation of PbS (lead sulfide) precipitates. Different from Cas9, we demonstrated that the crRNA transcribed from a standard type II RNA promoter was sufficient to guide Cpf1 endonuclease activity. Furthermore, modification of the crRNA with 3′ polyUs facilitates the faster maturation and folding of crRNA and improve the genome editing efficiency. We also achieved multiplexed genome editing, and the editing efficiency reached 75%–83% for duplex genomic targets (CAN1-URA3 and CAN1-MET25) and 41.7% for triplex genomic targets (CAN1-URA3-MET25). Taken together, this work expands the genome-editing toolbox for oleaginous yeast species and may accelerate our ability to engineer oleaginous yeast for both biotechnological and biomedical applications.
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spelling pubmed-69067112019-12-20 CRISPR-Cas12a/Cpf1-assisted precise, efficient and multiplexed genome-editing in Yarrowia lipolytica Yang, Zhiliang Edwards, Harley Xu, Peng Metab Eng Commun Article CRISPR-Cas9 has been widely adopted as the basic toolkit for precise genome-editing and engineering in various organisms. Alternative to Cas9, Cas12 or Cpf1 uses a simple crRNA as a guide and expands the protospacer adjacent motif (PAM) sequence to TTTN. This unique PAM sequence of Cpf1 may significantly increase the on-target editing efficiency due to lower chance of Cpf1 misreading the PAMs on a high GC genome. To demonstrate the utility of CRISPR-Cpf1, we have optimized the CRISPR-Cpf1 system and achieved high-editing efficiency for two counter-selectable markers in the industrially-relevant oleaginous yeast Yarrowia lipolytica: arginine permease (93% for CAN1) and orotidine 5′-phosphate decarboxylase (~96% for URA3). Both mutations were validated by indel mutation sequencing. For the first time, we further expanded this toolkit to edit three sulfur house-keeping genetic markers (40%–75% for MET2, MET6 and MET25), which confers yeast distinct colony color changes due to the formation of PbS (lead sulfide) precipitates. Different from Cas9, we demonstrated that the crRNA transcribed from a standard type II RNA promoter was sufficient to guide Cpf1 endonuclease activity. Furthermore, modification of the crRNA with 3′ polyUs facilitates the faster maturation and folding of crRNA and improve the genome editing efficiency. We also achieved multiplexed genome editing, and the editing efficiency reached 75%–83% for duplex genomic targets (CAN1-URA3 and CAN1-MET25) and 41.7% for triplex genomic targets (CAN1-URA3-MET25). Taken together, this work expands the genome-editing toolbox for oleaginous yeast species and may accelerate our ability to engineer oleaginous yeast for both biotechnological and biomedical applications. Elsevier 2019-11-22 /pmc/articles/PMC6906711/ /pubmed/31867213 http://dx.doi.org/10.1016/j.mec.2019.e00112 Text en © 2019 The Authors http://creativecommons.org/licenses/by/4.0/ This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).
spellingShingle Article
Yang, Zhiliang
Edwards, Harley
Xu, Peng
CRISPR-Cas12a/Cpf1-assisted precise, efficient and multiplexed genome-editing in Yarrowia lipolytica
title CRISPR-Cas12a/Cpf1-assisted precise, efficient and multiplexed genome-editing in Yarrowia lipolytica
title_full CRISPR-Cas12a/Cpf1-assisted precise, efficient and multiplexed genome-editing in Yarrowia lipolytica
title_fullStr CRISPR-Cas12a/Cpf1-assisted precise, efficient and multiplexed genome-editing in Yarrowia lipolytica
title_full_unstemmed CRISPR-Cas12a/Cpf1-assisted precise, efficient and multiplexed genome-editing in Yarrowia lipolytica
title_short CRISPR-Cas12a/Cpf1-assisted precise, efficient and multiplexed genome-editing in Yarrowia lipolytica
title_sort crispr-cas12a/cpf1-assisted precise, efficient and multiplexed genome-editing in yarrowia lipolytica
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6906711/
https://www.ncbi.nlm.nih.gov/pubmed/31867213
http://dx.doi.org/10.1016/j.mec.2019.e00112
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